Host neutralizing responses and pathogenesis of hepatitis C virus infection†

The hepatitis C virus (HCV) is a small enveloped positive-strand RNA virus that belongs to the genus Hepacivirus of the Flaviviridae family.1 Spontaneous viral clearance occurs in approximately 20% to 30% of acutely infected individuals and results in resolution of infection without sequelae. The majority of infected individuals, however, develop chronic hepatitis that may progress to liver cirrhosis and hepatocellular carcinoma.2 After infection, HCV starts to replicate in the host's hepatocytes and HCV RNA becomes detectable in the serum within 1 to 3 weeks.3 It is believed that the type and strength of the host's immune responses during the acute phase of HCV infection determines the outcome of infection. Although the role of CD4 and CD8 T cells in clearing HCV infection is widely accepted, the role antibodies play in HCV clearance is a matter of debate. Determining the relative contribution of antibodies to HCV clearance is difficult due to the lack of a convenient in vitro model system for evaluating the neutralizing potential of anti-HCV antibodies. The recent development of sensitive and robust neutralization assays based on HCV pseudotyped particles4-6 has enabled several investigators to study the role of neutralizing antibodies in acute and chronic HCV infection. HCV pseudotyped particles (HCVpp) consist of unmodified HCV envelope glycoproteins assembled onto retroviral6 or lentiviral5 core particles. HCVpp infect human hepatoma cell lines and hepatocytes in an HCV envelope protein–dependent manner. The presence of a green fluorescent protein or luciferase marker gene packaged within these HCVpp allows reliable and fast determination of antibody-mediated neutralization of infection. Another recently developed approach to study virus neutralization consists of recombinant cell culture–derived HCV (HCVcc) infecting human hepatoma cell lines.7-9 Mechanisms of viral entry and neutralization in vitro appear to be similar between HCVpp and HCVcc infection.10-13 Interestingly, two studies demonstrated that in vitro neutralization in the HCVpp model system correlated with neutralization of serum-derived HCV4, 14 and most recent results suggest that antibody-mediated neutralization of HCVpp appears to correlate with neutralization of HCV in the human liver uPA-SCID mouse in vivo.15 Although differences in the export pathway of HCVpp and native HCV may result in consequences for the properties of produced viral envelope,16, 17 the HCVpp system has been used by most investigators because it is characterized by a high robustness and the ability to perform high-throughput assays, allowing the quantification of virus neutralization of a large number of patient samples. The vast majority of antibodies induced in the course of HCV infection have no antiviral activity; they may be elicited by intracellular, degraded, or incompletely processed proteins released from dying cells or be directed against epitopes that do not play any role in the virus entry process.18, 19 Antibodies directed against the viral envelope proteins may prevent or control viral infection if they are directed against epitopes implicated in virus entry. These antibodies exhibit antiviral activity in neutralization assays in vitro and are thus termed neutralizing antibodies. Neutralization by antibodies may be mediated by several different mechanisms.18, 20 Neutralizing antibodies may directly block attachment of the virus to the host cell and thus inhibit dissemination of infection. In addition, neutralizing antibodies may interfere with postbinding steps such as entry of the virus into the host cell as well as transcription of the viral genome. Moreover, neutralizing antibodies may act as opsonins in enhancing phagocytosis of virus particles, thereby decreasing viral load. They may be classified as isolate-specific or cross-neutralizing depending on their ability to neutralize only the autologous virus (a defined viral strain present in the patient of interest) or heterologous viral strains (strains different from the autologous strain, usually obtained from individuals different from the patient of interest). Anti-HCV antibodies become detectable 4 to 14 weeks after infection3, 21 (Fig. 1). These antibodies target a wide variety of epitopes of both structural and nonstructural proteins. Early studies pointed to a role for antibodies in the control of HCV infection: HCV-infected patients with primary antibody deficiencies have been reported to have accelerated rates of disease progression, though alterations in T cell function may also have contributed to rapid disease progression in these patients.22, 23 In addition, passive protection against HCV has been demonstrated in different patient cohorts, including patients undergoing liver transplantation for HCV-related and hepatitis B virus–related liver cirrhosis and receiving infusion of anti–hepatitis B surface antigen hyperimmune globulin containing anti-HCV antibodies,24 as well as patients that had received immunoglobulin preparations derived from HCV RNA-positive plasma but containing HCV-neutralizing antibodies.14 Despite these findings, the role of antibodies in HCV clearance has long been questioned, because studies have reported resolution of HCV infection in the absence of detectable anti-HCV antibodies in standard diagnostic testing25 and presence of anti-HCV antibodies after an initial HCV infection that did not prevent reinfection in polytransfused thalassemic children.26 Other studies involving either patients with posttransfusion hepatitis C, health care workers, or young intravenous drug users failed to show any clear association between the presence of neutralizing antibodies in the acute phase of infection and viral clearance.21, 27, 28 Moreover, a recent study showed a higher frequency of neutralizing antibodies during the acute phase of infection in individuals who subsequently developed chronic HCV infection.29 Neutralizing humoral and cellular immune responses in resolving and chronic HCV infection. Resolving HCV infection is associated with rapid induction of virus-neutralizing antibodies as well as strong multispecific T cell responses. However, elimination of HCV has also been documented in the absence of neutralizing antibodies. Chronic HCV infection is characterized by absent or low-titer neutralizing antibodies as well as weak and narrow T cell responses during the early phase of infection and high-titer cross-neutralizing antibodies unable to control circulating isolates arise during the chronic phase of infection. Overall, these studies suggest that viral clearance can occur in the absence of neutralizing antibodies. However, the heterogeneous patient cohorts, the absence of a well-defined viral inoculum, and the fact that the viral surrogate ligands used for neutralization assays were derived from different isolates than the virus present in the infected individuals might have precluded the detection of isolate-specific neutralizing antibodies in the HCV-infected individuals analyzed in these studies. Most recently, studies using a well-defined viral inoculum and autologous surrogate ligands enabled the study of the role of isolate-specific neutralizing antibodies for control of HCV infection in humans. Two studies have demonstrated that neutralizing antibodies are induced in the early phase of infection by patients who subsequently clear the virus30 or control viral infection.31 A study involving hemodialysis patients with nosocomial acquired HCV infection that were followed over 6 months demonstrated that viral load appears to correlate with the presence of neutralizing antibodies during the acute phase of infection; emergence of strong neutralizing responses correlated with a decrease in viremia and control of HCV replication, whereas absence of a neutralizing response was associated with persistent high viremia and failure to control HCV infection.31 Moreover, in a well-characterized single-source outbreak of hepatitis C in young, healthy pregnant women who were accidentally exposed to HCV and followed up for over 25 years, viral clearance was associated in the majority of patients with the rapid induction of high-titer and cross-neutralizing antibodies in the acute phase. In contrast, chronic HCV infection was characterized by a nonexistent or reduced capacity to neutralize the transmitted virus as well as heterologous viruses in the early phase of infection.30 However, it is interesting to note that some patients (2 out of 17) were able to clear HCV infection without detection of neutralizing antibodies during the early phase of infection.30 These results suggest that a strong early broad neutralizing antibody response may contribute to control of HCV in the acute phase of infection and assist cellular immune responses in viral clearance but also underscore that viral clearance can occur in the absence of neutralizing antibodies. Both viral isolate as well as host-specific factors may have favored the induction of a potent neutralizing response in this cohort. Interestingly, the association between high-titer neutralizing antibody responses in the acute phase of HCV infection and viral clearance has been confirmed most recently in another group of HCV-infected individuals with intravenous drug abuse where clearance subjects developed high-titer neutralizing antibodies during acute infection, whereas the majority of individuals developing chronic infection had low titer or absent neutralizing antibodies during acute infection (S. C. Ray, personal communication, 2007). Taken together, these studies suggest that rapid induction of neutralizing responses may assist in control of HCV in the early phase of infection. HCV-specific T lymphocytes appear 5 to 9 weeks after infection (Fig. 1) and play an important role in both viral control and liver injury. CD4+ T cells have major regulatory functions, because they help CD8+ T cells eliminate infected cells and help B cells generate antibodies. Similar to studies showing an association between strong neutralizing responses during the first months after contamination and control of infection,30, 31 it has been demonstrated that patients who spontaneously clear HCV infection mount vigorous multi–epitope-specific CD4+ and CD8+ T cell responses.32-35 In line with these results, two recent studies point to a prominent role of CD4+ T cells in the control of human HCV infection.29, 36 It is interesting to note that the cellular immune responses persist for many years after elimination of the virus,37 whereas neutralizing antibody responses become weak or are lost after viral clearance30, 37 (Fig. 1). However, waning neutralizing antibody responses may not necessarily enhance susceptibility to new infection, because recall responses may exist as shown for host responses to other viral infections.19 Taken together, these studies suggest that clearance of HCV may be mediated by an orchestrated interplay of cellular and neutralizing immune responses. This is in line with recent studies demonstrating that immune control of other poorly cytopathic viruses, such as lymphocytic choriomeningitis virus or simian immunodeficiency virus/human immunodeficiency virus (HIV), requires a collaboration of both neutralizing antibodies and antiviral cellular responses.19, 38 CLDN1, claudin-1; HCV, hepatitis C virus; HCVcc, cell culture–derived hepatitis C virus; HCVpp, hepatitis C virus pseudotyped particles; HDL, high-density lipoprotein; HVR1, hypervariable region 1; LDL, low-density lipoprotein; SR-BI, scavenger receptor class B type I; VLDL, very low-density lipoprotein. In the majority of individuals, the immune system fails to eliminate HCV during the first 6 months after contamination, and HCV infection persists. HCV replication, estimated by HCV RNA peripheral blood levels, seems to remain relatively stable in these individuals (Fig. 1). Persistent HCV infection is characterized by the induction of HCV-specific antibodies that are directed against both structural and nonstructural proteins and are able to cross-neutralize heterologous viruses or quasispecies that arise in the course of infection.4, 27, 28, 30, 37, 39, 40 A kinetic study of neutralizing antibody responses against the viral inoculum in a single-source outbreak of HCV infection showed that isolate-specific as well as cross-neutralizing antibodies emerged during the chronic phase of infection.30 Paradoxically, these “neutralizing” antibodies induced during chronic HCV infection are not able to clear the virus. Additionally, an alternative kinetic study of acute monoinfected patients revealed that high-density lipoprotein (HDL) is a serum factor that impairs the efficiency of the cross-neutralizing antibodies that are present during the acute phase of the disease.31, 41 Altogether, these results indicate that HCV has evolved mechanisms that counteract the impact of the humoral response during both the acute and the chronic phase of the disease. In line with these data, it has been shown that HCV genome complexity is associated with the inability to clear HCV infection and development of chronic disease; whereas resolving hepatitis C was associated with a relatively stable pool of viral variants, progression into chronic infection correlated with diversification of the quasispecies population.42-44 Taken together, these studies demonstrate that the host neutralizing responses are not able to control the circulating pool of viruses during chronic infection. Using various HCV model systems (for review, see Barth et al.45) rapid progress has been made in understanding viral escape from antibody-mediated neutralization. A prerequisite of the understanding of these mechanisms has been the identification of various host entry factors mediating the first steps of viral infection (for review, see Barth et al.45 and Bartosch and Cosset46): binding and entry of HCV is believed to be a multistep process involving several host cell surface molecules such as CD81,47 scavenger receptor class B type I (SR-BI),48 the glycosaminoglycan heparan sulfate,49, 50 claudin-1 (CLDN1),51 and the low-density lipoprotein (LDL) receptor52 (Fig. 2). Most recently, it has been shown that in addition to CLDN1, two other members of the claudin family, CLDN6 and CLDN9, may also function as HCV coreceptors.53 Kinetic studies have recently demonstrated that heparan sulfate predominantly plays a role during HCV attachment,54 whereas CD81,54-56 SR-BI,57-59 and CLDN151 promote HCV entry into target cells during postbinding steps. HCV is most certainly internalized in a clathrin-dependent manner,60-62 and HCV genome delivery into the host cell cytosol prior to HCV replication is pH-dependent (Fig. 2).60, 63 By analogy to other viral infections, antibodies neutralizing HCV may render virions noninfectious by interfering with different steps of the viral life cycle such as binding, entry, or fusion. Several viral epitopes targeted by neutralizing antibodies elicited by HCV-infected individuals have already been identified (reviewed by Zeisel et al.64). However, the mechanisms of antibody-mediated HCV neutralization remain elusive. Most recently, using a panel of monoclonal anti-HCV envelope antibodies, it was shown that antibodies may neutralize HCV during various steps of HCV entry, including binding and postbinding events. Moreover, polyclonal anti-HCV immunoglobulin purified from HCV-infected patients was able to target different steps of viral entry including membrane fusion (A. Haberstroh, E. K. Schnober, M. B. Zeisel, P. Carolla, H. Barth, B. Gissler, H. E. E. H. C. M. M. and Mechanisms of viral escape from antibody-mediated neutralization during HCV entry. HCV binding and entry is a multistep after attachment of viral envelope glycoproteins and to host cell surface HCV is internalized into the host cell in the to fusion between viral and and to the of the viral genome into the from et escape from antibody-mediated neutralization has been shown to occur including the of antibodies binding of neutralizing antibodies to viral association of HCV with including and VLDL, that may neutralizing the interplay of HCV with and that is able to protection from neutralizing the of neutralizing epitopes by of of HCV envelope and of the virus. heparan low-density lipoprotein not identified host factors for HCV binding and entry. escape from antibody-mediated neutralization has been shown to occur on several levels, including the high of the HCV genome and induction of cross-neutralizing the induction of antibodies interfering with neutralizing the association of HCV with serum factors such as and very low-density lipoprotein the interplay of HCV glycoproteins and with the of neutralizing epitopes by of defined of envelope and of the virus. a of different mechanisms (for of neutralizing epitopes or receptor binding by or by as for viruses such as may also to HCV an viral this virus as a pool of but (a in infected The immune system is believed to on these viral variants, the emergence of T and B cell escape the by and of a well-characterized chronic HCV patient who was infected with HCV and was the of HCV strains and new into the of induction of neutralizing antibodies and viral escape from humoral responses. By using HCVpp envelope glycoproteins of HCV present different during acute and chronic infection, the investigators demonstrated that neutralization of heterologous strains did not neutralization of the viral present in the serum the of blood In a the “neutralizing” antibodies of this patient were able to neutralize HCV strains that had been present several months or years but not the present or viral Several in defined B cell epitopes in the HCV envelope be identified and may escape from neutralizing antibody responses. This escape from neutralizing antibodies in the course of HCV infection has been recently confirmed by another study of neutralizing antibody responses in acute HCV infection (S. C. Ray, personal communication, 2007). Most recently, it has been shown that anti-HCV antibody containing immunoglobulin preparations interfering antibodies in addition to neutralizing of epitopes targeted by these revealed two HCV envelope but only was in HCV neutralization. In contrast, binding of antibodies to the other viral neutralization by the neutralizing These suggest that HCV both neutralizing and antibodies, the able to interfere with the function of neutralizing antibodies, thereby allowing escape from the host's humoral responses. from antibody-mediated virus neutralization may also occur of epitopes targeted by neutralizing antibodies the association of HCV with It has been demonstrated that HCV in heterogeneous in human serum and may be associated with VLDL, LDL, and These viral particles may human hepatoma cells their associated lipoprotein in an HCV envelope The association of HCV with these may thus the virus from neutralizing antibodies the HCV envelope studies using the recombinant HCVcc associated with may study of these mechanisms in the In to and VLDL, was identified as a serum factor enhancing in vitro HCV This has been shown to be mediated by in HDL, and by the properties of an the interplay of HCV glycoproteins with and is able to protection from cross-neutralizing antibodies present in of acute and chronic HCV-infected The hypervariable region 1 of the HCV envelope has been associated with an inability to clear the to play a major role in this In of HCVpp glycoproteins was not by of this region in HCVpp that were not or by serum from HCV-infected individuals was able to the HCV neutralizing ability of antibodies. Moreover, antibodies directed against various epitopes of HCV envelope were to neutralize HCVpp in the presence of HDL, whereas this to antibody-mediated neutralization was in HCVpp or in where the activity of was These results suggest that the between the viral HVR1, HDL, and a to HCV from neutralization by antibodies targeted the of HCV to the host's neutralizing responses is of epitopes that might be targeted by neutralizing antibodies In HCV envelope glycoproteins are and this may their Two recent studies have shown that of several from HCV envelope glycoproteins antibody-mediated neutralization of HCV and showed that 3 on HCV envelope protein reduced the of HCVpp to antibody-mediated neutralization. These also of to binding of these HCVpp sensitive to neutralization by serum from HCV-infected This also of HCVpp with Similar results have been obtained by and who reported that 4 in the of reduced HCVpp to neutralization. Two out of these 4 also These suggest that several the binding a major target for neutralizing antibodies. of these to the impact of neutralizing antibodies targeted to these the virus also to the ability to to in to target This may has been shown for these on HCV are may also antibody response to the HCV envelope is a studies that of this viral protein can be by different Taken together, these studies show that of HCV envelope glycoproteins can protection against virus-neutralizing antibodies. HCV may also escape from neutralizing antibodies by infecting cells by In the majority of viruses infect cells after the of viral particles that are released from infected cells and cells by a These viral particles are to neutralization by antibodies. viruses may also to infect thereby potential with neutralizing antibodies. This has been for various viruses, including human T cell virus type and viruses (reviewed by and for HCV has been and H. personal communication, 2007). and of HCV-infected cells with cells in the presence of neutralizing antibodies. A higher of fluorescent HCV-infected cells be demonstrated cells and target cells were to in with cells in by a using human cells as well as cells with a suggest that HCV is Moreover, because antibodies may also be mediated in by Interestingly, human hepatoma cells that are to infection by HCV were sensitive to HCV infection in this model system in the presence of virus-neutralizing antibodies, that are also of was also demonstrated by and H. communication, who demonstrated and replication of HCVcc that are unable to target cells from the Taken together, these studies demonstrate that HCV might by two different and thereby neutralizing antibodies. The of escape mechanisms from immune responses be for the understanding of HCV as well as the development of and for control of HCV infection. Moreover, immune responses against HCV as in individuals spontaneously clearing infection might to such immune responses by from acute and chronic HCV-infected patients as well as studies in suggest that a be able to vigorous and multispecific B and T cell responses in to control viral infection and prevent progression to In recent years, the study of to HCV in intravenous drug users who HCV infection that protection from HCV might exist in if against various viral strains might be difficult to progression into chronic HCV infection and thus development of cirrhosis and hepatocellular may be by a or Moreover, studies have shown that a broad against HCV can be induced as that had a first infection were able to clear HCV with or heterologous studies in showed that different are able to protection of the host against different viral strains and reduced persistent Interestingly, it appears that both cellular and humoral immune responses might be (reviewed by and In addition, of on HCV envelope glycoproteins to and cellular and humoral immune response in mouse the of HCV escape mechanisms to Most recently, human monoclonal cross-neutralizing antibodies directed against epitopes of HCV envelope or were These antibodies were able to block infection with heterologous HCV in the HCVpp and HCVcc model and two of were able to from with heterologous HCV in using the human mouse These results indicate that a against HCV may be passive in with or antiviral is another important to for health care and to prevent HCV reinfection during liver Chronic hepatitis C is a for liver liver transplantation in HCV-infected patients is followed by reinfection of the In addition, hepatitis C in patients is to The fact that of HCV infection the of immunoglobulin preparations derived from plasma from anti-HCV antibodies had been suggest that anti-HCV antibodies may prevent viral infection in Moreover, and demonstrated the of in the an immunoglobulin derived from the plasma of HCV-infected patients rapid clearance of viremia and development of acute infection in the with anti-HCV neutralizing antibodies might thus an to prevent liver This approach has already been used to prevent reinfection of liver in hepatitis B studies using anti-HCV antibodies in liver patients have not been to (reviewed by It has been that this might be due to antibody or frequency of It has also been that the presence of antibodies in some anti-HCV antibody containing immunoglobulin preparations may epitopes for neutralizing antibodies, thereby these preparations escape mechanisms of anti-HCV antibody containing immunoglobulin preparations with neutralizing antibodies by of interfering antibodies might to the efficiency of in HCV-infected the recent of human monoclonal antibodies cross-neutralizing HCV may an important for the development of against HCV Taken together, progress has been made in neutralizing responses in the course of HCV infection, and recent studies underscore the of neutralizing antibodies in HCV the understanding of the mechanisms of antibody-mediated virus neutralization the development of new and antiviral against HCV infection. The H. C. Ray, and for results, for and C. M. and M. for of the